Summary This application requests continued support for a long-standing P01 project on peripheral tolerance. This has been a highly productive P01 that provided mechanistic insight about clonal deletion and anergy. These discoveries were made through technical innovation in how to identify and manipulate self-antigen specific T cells using pMHC tetramers. Based on preliminary data suggesting that mice with normal microbial experience have strikingly altered immunity compared to specific pathogen free mice, we propose to focus in the next period on understanding tolerance in the face of diverse pathogen experience, which is the reality for wild animals and humans. The program includes five projects that will focus on distinct but complimentary aspects of tolerance: natural MHCII-bound self epitope discovery, polyclonal anergic cells, type I interferons, non- deletional CD8+ T cell tolerance, and hybrid self-peptides. The five investigatorial teams propose a strongly synergistic plan to build a comprehensive understanding of physiologic T cell tolerance mechanisms that operate in the face of normal microbial experience, and to continue to develop tools that have a broad impact as we do so. The project includes three core facilities to enhance research productivity: 1) a mouse core to facilitate and standardize research on mice bearing a diverse pathogen load, 2) a human tissue core to facilitate the immunological analysis of multiple human tissues, not merely peripheral blood, and 3) an administrative core to carry out activities that enhance the synergy and productivity of the group.